WO2024253424A1 - Anticorps humanisé se liant de manière spécifique à la protéine trem2 humaine et son utilisation - Google Patents

Anticorps humanisé se liant de manière spécifique à la protéine trem2 humaine et son utilisation Download PDF

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WO2024253424A1
WO2024253424A1 PCT/KR2024/007703 KR2024007703W WO2024253424A1 WO 2024253424 A1 WO2024253424 A1 WO 2024253424A1 KR 2024007703 W KR2024007703 W KR 2024007703W WO 2024253424 A1 WO2024253424 A1 WO 2024253424A1
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amino acid
acid sequence
sequence represented
heavy chain
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Korean (ko)
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임현호
황준모
박소라
최소영
이세라
김상길
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Daegu Gyeongbuk Institute of Science and Technology
Osong Medical Innovation Foundation
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Daegu Gyeongbuk Institute of Science and Technology
Osong Medical Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a humanized antibody that specifically binds to human TREM2 (Triggering receptor expressed on myeloid cells 2) protein and uses thereof.
  • human TREM2 Triggering receptor expressed on myeloid cells 2
  • TREM2 Triggering receptor expressed on myeloid cells 2 is a receptor that transmits signals through an adapter protein called DAP12. It is known to regulate myeloid cell function and suppresses inflammatory responses by inhibiting the expression of cytokines, confirming that TREM2 is involved in immune regulation.
  • the TREM2 protein is mainly present in microglia and has been found to play an important role in phagocytosis of dementia target factors, amyloid-beta, processing of damaged neurons, and maintaining immune cell homeostasis.
  • TREM2 is a cell membrane protein, so it is difficult to purify it in its original intact form within the cell while maintaining its original function within the cell as much as possible, and antibodies that can specifically detect TREM2 protein are also insufficiently developed.
  • mice used to produce monoclonal antibodies.
  • non-human antibodies such as mouse-derived monoclonal antibodies are considered foreign antigens in the human body, so they induce an immune response and have a short half-life, which limits their therapeutic effect.
  • humanized antibodies have been developed in which only the antigen-binding portion of the antibody is replaced with a human antibody.
  • the currently used method of replacing a mouse antibody with a humanized antibody is to select the most similar human antibody gene for the antibody to be replaced, and replace only the CDR portion of the mouse antibody with the human antibody CDR position using a method called CDR grafting.
  • Such humanized antibodies have the advantage of reducing the immune response in the human body because most of the genes are humanized.
  • the purpose of the present invention is to provide a humanized antibody or an antigen-binding fragment thereof that specifically binds to human TREM2 protein.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the humanized antibody or an antigen-binding fragment thereof, a recombinant expression vector comprising the nucleic acid molecule, and an isolated cell transformed with the recombinant expression vector.
  • Another object of the present invention is to provide a composition for detecting human TREM2 antigen comprising the humanized antibody or an antigen-binding fragment thereof as an effective ingredient.
  • Another object of the present invention is to provide a composition for diagnosing Alzheimer's disease comprising the humanized antibody or an antigen-binding fragment thereof as an effective ingredient.
  • the present invention provides a heavy chain variable region comprising any one heavy chain FR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence represented by SEQ ID NO: 8, an amino acid sequence represented by SEQ ID NO: 9, and an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 10, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 5, an amino acid sequence represented by SEQ ID NO: 11, and an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR1 selected from the group consisting
  • the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds to human TREM2 protein, comprising: a light chain variable region comprising a light chain FR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 22, and an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 23, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 20, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 21.
  • a light chain variable region comprising a light chain FR1 selected from the group consisting of an amino acid
  • the present invention also provides a nucleic acid molecule encoding the humanized antibody or an antigen-binding fragment thereof.
  • the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
  • the present invention provides an isolated cell transformed with the recombinant expression vector.
  • the present invention provides a composition for detecting human TREM2 antigen comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing Alzheimer's disease comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention relates to a humanized antibody that specifically binds to human TREM2 protein and a use thereof, and a humanized antibody was produced by grafting complementarity determining regions (CDRs) corresponding to antigen-binding sites of a variable region of a mouse antibody onto a human germline-derived sequence using a CDR grafting technique, thereby securing a humanized antibody that specifically binds to TREM2.
  • CDRs complementarity determining regions
  • the obtained humanized antibody was confirmed to have antigen-binding ability and biological activity, and an IgG1-type chimeric antibody was produced by converting it into IgG2 and IgG4 types, and its antigen-binding ability and biological activity were confirmed.
  • the present invention has high affinity for TREM2, it is possible to develop an antibody-based diagnostic system capable of diagnosing TREM2 as a biomarker for various Alzheimer's dementias.
  • the existing Alzheimer's treatment drugs are symptom-relieving drugs that temporarily regulate neurotransmitters, it is thought that the development of an antibody targeting TREM2 will lead to the development of a promising new drug with a practical therapeutic effect.
  • Figure 1 shows the SE-HPLC analysis results of chimeric antibody proteins.
  • Figure 2 shows the results of SDS-PAGE gel analysis and SE-HPLC analysis of humanized antibody proteins.
  • Figure 3 shows the results of evaluating the binding affinity of TREM2-binding antibodies to human TREM2.
  • Figure 4 shows the results of the Humanness score analysis of humanized antibodies.
  • Figure 5 shows the results of in silico immunogenicity prediction analysis of humanized antibodies.
  • Figure 6 shows the results of efficacy verification of chimeric and humanized antibodies.
  • Figure 7 shows the results of efficacy verification of chimeric antibodies converted to IgG2 and IgG4 forms.
  • the present invention relates to a heavy chain variable region comprising: a heavy chain FR1 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence represented by SEQ ID NO: 8, an amino acid sequence represented by SEQ ID NO: 9, and an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 3, an amino acid sequence represented by SEQ ID NO: 10, and an amino acid sequence represented by SEQ ID NO: 13, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 selected from the group consisting of an amino acid sequence represented by SEQ ID NO: 5, an amino acid sequence represented by SEQ ID NO: 11, and an amino acid sequence represented by SEQ ID NO: 14, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 comprising an
  • the present invention provides a humanized antibody or an antigen-binding fragment thereof that specifically binds to human TREM2 (Triggering receptor expressed on myeloid cells 2) protein, wherein the light chain variable region comprises: a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 15, an amino acid sequence represented by SEQ ID NO: 22, and an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 19 or SEQ ID NO: 23, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 20, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 21.
  • the light chain variable region comprises: a light chain FR1 comprising an amino acid sequence
  • the humanized antibody or antigen-binding fragment thereof that specifically binds to the human TREM2 protein comprises: 1) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 3, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7, 2) a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 8, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by an amino acid
  • a heavy chain variable region comprising a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7, 3) a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 9, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino acid sequence represented by SEQ ID NO: 10, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain FR3 consisting of an amino acid sequence represented by SEQ ID NO: 11, a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6, and a heavy chain FR4 consisting of an amino acid sequence represented by SEQ ID NO: 7, and 4) a heavy chain variable region comprising a heavy chain FR1 consisting of an amino acid sequence represented by SEQ ID NO: 12, a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 2, a heavy chain FR2 consisting of an amino
  • a humanized antibody or an antigen-binding fragment thereof characterized in that it comprises any one light chain variable region selected from the group consisting of a light chain FR4 comprising a sequence and 3) a light chain FR1 comprising an amino acid sequence represented by SEQ ID NO: 24, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 16, a light chain FR2 comprising an amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 18, a light chain FR3 comprising an amino acid sequence represented by SEQ ID NO: 23, a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 20, and a light chain FR4 comprising an amino acid sequence represented by SEQ ID NO: 21, but is not limited thereto.
  • the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and examples thereof may include a monoclonal antibody, a polyclonal antibody, a full-length antibody, and an antibody fragment.
  • the term “antibody” may include a bivalent or dual specific molecule (e.g., a bispecific antibody), a diabody, a triabody, or a tetrabody.
  • the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibodies exhibit single binding affinity and binding specificity for a specific epitope, unlike polyclonal antibodies that can bind to multiple epitopes.
  • the term “full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to the heavy chain by a disulfide bond.
  • the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types and has gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1), and alpha 2 ( ⁇ 2) as subclasses.
  • the light chain constant region has kappa ( ⁇ ) and lambda ( ⁇ ) types.
  • IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.
  • chimeric antibody in the present invention is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and is an antibody with a greatly improved immune response compared to a mouse antibody.
  • humanized antibody means an antibody in which all or part of the CDR sequence of a mouse monoclonal antibody is grafted onto a human antibody, and for example, the CDRs of a mouse monoclonal antibody are recombined with FRs derived from a human antibody to produce a humanized variable region, which is then recombined with the constant region of a desired human antibody to produce the antibody.
  • FRs derived from a human antibody
  • humanized variable region which is then recombined with the constant region of a desired human antibody to produce the antibody.
  • a humanized antibody is an antibody whose amino acid sequence is altered, and the amino acid sequence of the antibody can be reconstructed according to the desired purpose.
  • variable region modifications in the variable region are performed to increase the binding ability and affinity of the antigen, whereas modifications in the constant region are performed to increase intracellular functions such as fixation of complement, interaction with the membrane, and functions of other effectors.
  • the term “heavy chain” may include both a full-length heavy chain and fragments thereof, comprising a variable region VH and three constant regions CH1, CH2 and CH3, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • the term “light chain” in the present invention may include both a full-length light chain and fragments thereof, comprising a variable region VL and a constant region CL, which include an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
  • fragment In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody.
  • exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.
  • the antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the antibodies described herein, but also biological equivalents thereof, to the extent that they can exhibit the ability to specifically bind to human TREM2.
  • additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody.
  • Such changes may include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of the antibody.
  • Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Accordingly, based on this advantage, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
  • the present invention also provides a nucleic acid molecule encoding the humanized antibody or an antigen-binding fragment thereof.
  • nucleic acid molecule has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified.
  • sequence of a nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.
  • the present invention provides a recombinant expression vector comprising the nucleic acid molecule.
  • vector means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).
  • the “expression vector” means a recombinant DNA molecule including a target coding sequence and an appropriate nucleic acid sequence essential for expressing the coding sequence operably linked in a specific host organism.
  • the expression vector may preferably include one or more selectable markers.
  • the markers are nucleic acid sequences having characteristics that can be selected, typically by a chemical method, and include all genes that can distinguish transformed cells from non-transformed cells. Examples thereof include, but are not limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol, and can be appropriately selected by those skilled in the art.
  • any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the promoter and enhancer of CMV, the LTR of retroviruses, the lac system, the trp system, the TAC or TRC systems, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of such phosphatases, e.g. Pho5, the promoter of the yeast alpha-mating system, and any other sequence of structure and inducibility known to control the expression of genes of prokaryotes or eukaryotes or their viruses, and various combinations thereof.
  • the vector expressing the antibody of the present invention can be a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation.
  • Co-transformation is a method in which each vector DNA encoding the light chain and the heavy chain is simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected.
  • Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), to finally select cells expressing both the light chain and the heavy chain.
  • the present invention provides an isolated cell transformed with a recombinant expression vector.
  • the cell capable of stably and continuously cloning and expressing the vector of the present invention can be any host cell known in the art, including but not limited to, prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylocus carnosus).
  • prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylocus carnosus).
  • the culture of transformed cells can be carried out according to appropriate media and culture conditions known in the relevant technical field.
  • Such culture process can be easily adjusted and used by a person skilled in the art according to the selected strain.
  • Cell culture is divided into suspension culture and attachment culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method.
  • the medium used for culture must appropriately satisfy the requirements of a specific strain.
  • the present invention provides a composition for detecting human TREM2 antigen comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.
  • the present invention provides a composition for diagnosing Alzheimer's disease comprising the humanized antibody or an antigen-binding fragment thereof as an active ingredient.
  • variable regions of the heavy and light chains of mouse antibodies C2 and F2 were cloned into the animal cell expression vector pcDNATM 3.4 TOPO vector (ThermoFisher Scientific) together with the human IgG1 heavy chain constant region (P01857), IgG2 heavy chain constant region (P01859), IgG4 heavy chain constant region introducing the S228P mutation (P01861), and human kappa light chain constant region (P01834).
  • each plasmid DNA was transfected into Expi293 as described in the Thermo Fisher manufacturer's protocol (ExpiFectamine 293 transfection kit, ThermoFisher Scientific). Briefly, 6x106 cells/ml Expi293 cells were transfected with DNA and ExpiFectamine complexes and cultured at 37°C, 8% CO2 , and the expression enhancer was added 24 h later. On the second day after transfection, the cells were cultured at 32°C for 6 days, and the culture supernatant containing TREM2-binding antibody proteins was collected.
  • a Protein A affinity chromatography column (GE Healthcare) was equilibrated with PBS (pH 7.4). The culture supernatant containing each TREM2-binding antibody protein was filtered through a 0.2 ⁇ m filter and loaded onto the Protein A affinity chromatography column. After washing the column with PBS (pH 7.4), the TREM2-binding antibody protein was eluted with a 100 mM glycine (pH 3.4) solution. The eluate was neutralized by adding 1 M Tris solution and the buffer was exchanged with PBS.
  • the purified TREM2-binding antibody was confirmed by SDS-PAGE gel analysis (Invitrogen) and SE-HPLC analysis, and the protein concentration was quantified using a Nanodrop 2000C spectrophotometer (Thermo Scientific).
  • the SE-HPLC analysis results confirmed that the C2 IgG1, IgG2, and IgG4 chimeric antibodies were each more than 90% pure (Fig. 1).
  • the amino acid sequences of the heavy and light chain variable regions of the mouse monoclonal antibody C2 were compared and analyzed with the amino acid sequences of human germline antibodies in the IMGT database, and the heavy chain genes IGHV3-33, IGHV3-30, IGHV4-34, IGHV4-4, IGHJ4 and the light chain genes IGKV2-18, IGKV2-28, IGKV2-29 of the human antibody variable regions with high similarity were selected.
  • the CDR of the mouse antibody was grafted onto an IgG1 human antibody, and some amino acid residues of the humanized heavy chain FR (Framework) were substituted with the sequence of the mouse antibody.
  • a total of 12 humanized antibodies were produced by combining four heavy chains and three light chains (Tables 1 and 2).
  • humanized antibodies Twelve types were produced using human germline-based CDR grafting technology and transiently expressed in Expi293 cells.
  • the production method of humanized antibody proteins was performed in the same manner as the production method of chimeric antibodies.
  • the humanized antibodies were confirmed to have a purity of more than 95% (Fig. 2).
  • the binding affinity to the antigen was measured as the K D value for each antibody using Biacore.
  • the system and sample buffer used were HBS-EP (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% Polysorbate 20 (v/v)).
  • the antibody was immobilized on the surface of the CM5 chip using the amine coupling procedure according to the manufacturer's protocol of the human antibody capture kit (Cytiva).
  • the antigen protein diluted in HBS-EP buffer was flowed through the CM5 chip with the antibody immobilized at a flow rate of 30 ⁇ L/min for 2 minutes for binding and 5 minutes for dissociation.
  • the antigen protein bound to the antibody was washed by reacting with 3 M magnesium chloride for 60 seconds.
  • the sensorgram of the antibody was analyzed using Biacore evaluation software. As a result, humanized antibodies showing a similar level of binding ability compared to the binding ability of the chimeric antibody chiC2 (K D : 6.28E-10 M) were selected (Fig. 3).
  • the humanness scores of the light and heavy chains of mouse antibodies and the light and heavy chains of humanized antibodies were confirmed through the T20 score.
  • the humanized sequence increased both the T20 score and the humanness score was improved compared to the heavy and light chains of the mouse antibodies (Fig. 4).
  • TREM2 loses its function when the outer part of the cell membrane is cleaved by sheddase such as ADAM10/17, and at the same time, the amount of cleaved soluble TREM2 (sTREM2) present in the cell culture medium increases.
  • the antibody specified in the present invention can inhibit shedding by binding to TREM2 exposed to the outer part of the cell membrane, and this protective effect was evaluated as a decrease in the amount of sTREM2 present in the culture medium and internal TREM2 (iTREM2) present in the cell, and an increase in the protein amount of membrane TREM2 (mTREM2) (Fig. 6).
  • HEK2393T cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) were transfected with polyethylenimine (PEI) to express human TREM2-His and DAP12-V5. After culturing for 8 hours, the cells were replaced with fresh culture medium containing the control (C2 and human IgG produced from mouse) or each human antibody at a concentration of 20 ug/mL and further cultured for 16 hours.
  • pure culture medium and cell fraction samples total, internal, membrane fraction
  • the whole cell fraction was prepared by dissolving the cells obtained by centrifugation in the lysate (1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC)), rotating them in a refrigerator (4°C) for 1 hour, and then centrifuging them to remove the remaining debris to secure all the intracellular and cell membrane fraction proteins.
  • lysate 1% Triton X-100, 1X phosphate buffered saline (PBS), 1X protease inhibitor cocktail (PIC)
  • the intracellular fraction was prepared by dissolving the cells obtained by centrifugation in a storage solution (20 mM Tris, pH 7.0, 1 mM EDTA/EGTA, 1X PIC), keeping them on ice for 30 minutes to fill the inside of the cells with the buffer, and then freezing them in an ultra-low temperature freezer (-80°C) and thawing them to rupture the cells, followed by centrifugation to separate the intracellular fraction from the cell membrane fraction.
  • the proteins of the remaining cell membrane fraction were eluted from the cell membrane using the lysate, and then separated/secured from the debris through centrifugation.
  • PVDF polyvinylidene difluoride
  • the humanized antibodies ChiC2, HuC2-2, HuC2-5, and HuC2-9 described in the present invention showed excellent inhibitory effects compared to the shedding inhibition of mouse-derived antibody C2, and HuC2-6, HuC2-8, HuC2-10, and HuC2-10 showed effects at a similar level to the shedding inhibition of mouse-derived antibody C2 (One-Way ANOVA, Fisher test) (Table 3).
  • Antibody - IgG ChiC2 _ IgG1 ChiC2 _ IgG2 ChiC2 _ IgG4 C2 average 0.54 0.55 2.26 2.13 2.27 1.00 Standard error 0.10 0.09 0.22 0.03 0.24 0.01

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Abstract

La présente invention concerne un anticorps humanisé se liant de manière spécifique à une protéine TREM2 humaine et son utilisation. L'anticorps humanisé est préparé à l'aide d'une technologie de greffage CDR, une région déterminant la complémentarité (CDR) correspondant à un site de liaison à l'antigène d'une région variable d'anticorps de souris étant transplantée dans une séquence dérivée de lignée germinale humaine, ce qui permet de fixer l'anticorps humanisé se liant de manière spécifique à TREM2. L'anticorps humanisé obtenu a été confirmé pour la capacité de liaison à un antigène et à une activité biologique, et un anticorps chimérique sous la forme d'IgG 1 a été converti en formes d'IgG 2 et d'IgG 4 et produit, et confirmé pour la capacité de liaison à un antigène et à une activité biologique. Avec une affinité élevée pour TREM2, la présente invention peut développer un système de diagnostic à base d'anticorps capable de diagnostiquer TREM2 en tant que biomarqueur de diverses démence d'Alzheimer. De plus, étant donné que les agents thérapeutiques existants pour la maladie d'Alzheimer sont principalement des agents atténuant les symptômes dus à la régulation temporaire des neurotransmetteurs, il est considéré que le développement d'anticorps ciblant TREM2 peut conduire au développement de nouveaux médicaments prometteurs ayant des effets thérapeutiques importants.
PCT/KR2024/007703 2023-06-09 2024-06-05 Anticorps humanisé se liant de manière spécifique à la protéine trem2 humaine et son utilisation Pending WO2024253424A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20230074264 2023-06-09
KR10-2023-0074264 2023-06-09
KR1020240073132A KR20240174996A (ko) 2023-06-09 2024-06-04 인간 trem2 단백질에 특이적으로 결합하는 인간화 항체 및 이의 용도
KR10-2024-0073132 2024-06-04

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WO2024253424A1 true WO2024253424A1 (fr) 2024-12-12

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180068999A (ko) * 2015-10-06 2018-06-22 알렉터 엘엘씨 항-trem2 항체 및 그의 사용방법
KR20220084152A (ko) * 2019-11-22 2022-06-21 일라이 릴리 앤드 캄파니 Trem2 항체 및 그의 용도
KR20220131246A (ko) * 2020-01-13 2022-09-27 데날리 테라퓨틱스 인크. 항-trem2 항체 및 이의 사용 방법
US20230107639A1 (en) * 2014-08-08 2023-04-06 Alector Llc Anti-trem2 antibodies and methods of use thereof
US20230167174A1 (en) * 2018-10-15 2023-06-01 Novartis Ag Trem2 stabilizing antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230107639A1 (en) * 2014-08-08 2023-04-06 Alector Llc Anti-trem2 antibodies and methods of use thereof
KR20180068999A (ko) * 2015-10-06 2018-06-22 알렉터 엘엘씨 항-trem2 항체 및 그의 사용방법
US20230167174A1 (en) * 2018-10-15 2023-06-01 Novartis Ag Trem2 stabilizing antibodies
KR20220084152A (ko) * 2019-11-22 2022-06-21 일라이 릴리 앤드 캄파니 Trem2 항체 및 그의 용도
KR20220131246A (ko) * 2020-01-13 2022-09-27 데날리 테라퓨틱스 인크. 항-trem2 항체 및 이의 사용 방법

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